Effective, Rapid, and Small-Scale Bioconjugation and Purification of “Clicked” Small-Molecule DNA Oligonucleotide for Nucleic Acid Nanoparticle Functionalization
Nucleic acid-based therapeutics involves the conjugation of small molecule drugs to nucleic acid oligomers to surmount the challenge of solubility, and the inefficient delivery of these drug molecules into cells. “Click” chemistry has become popular conjugation approach due to its simplicity and hig...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-03-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/5/4797 |
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| author | Erwin Doe Hannah L. Hayth Ross Brumett Emil F. Khisamutdinov |
| author_facet | Erwin Doe Hannah L. Hayth Ross Brumett Emil F. Khisamutdinov |
| author_sort | Erwin Doe |
| collection | DOAJ |
| description | Nucleic acid-based therapeutics involves the conjugation of small molecule drugs to nucleic acid oligomers to surmount the challenge of solubility, and the inefficient delivery of these drug molecules into cells. “Click” chemistry has become popular conjugation approach due to its simplicity and high conjugation efficiency. However, the major drawback of the conjugation of oligonucleotides is the purification of the products, as traditionally used chromatography techniques are usually time-consuming and laborious, requiring copious quantities of materials. Herein, we introduce a simple and rapid purification methodology to separate the excess of unconjugated small molecules and toxic catalysts using a molecular weight cut-off (MWCO) centrifugation approach. As proof of concept, we deployed “click” chemistry to conjugate a Cy3-alkyne moiety to an azide-functionalized oligodeo-xynucleotide (ODN), as well as a coumarin azide to an alkyne-functionalized ODN. The calculated yields of the conjugated products were found to be 90.3 ± 0.4% and 86.0 ± 1.3% for the ODN-Cy3 and ODN-coumarin, respectively. Analysis of purified products by fluorescence spectroscopy and gel shift assays demonstrated a drastic amplitude of fluorescent intensity by multiple folds of the reporter molecules within DNA nanoparticles. This work is intended to demonstrate a small-scale, cost-effective, and robust approach to purifying ODN conjugates for nucleic acid nanotechnology applications. |
| first_indexed | 2024-03-11T07:21:33Z |
| format | Article |
| id | doaj.art-68bc76c81dda4851bc9f0bed992d02e6 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T07:21:33Z |
| publishDate | 2023-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-68bc76c81dda4851bc9f0bed992d02e62023-11-17T07:53:33ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-03-01245479710.3390/ijms24054797Effective, Rapid, and Small-Scale Bioconjugation and Purification of “Clicked” Small-Molecule DNA Oligonucleotide for Nucleic Acid Nanoparticle FunctionalizationErwin Doe0Hannah L. Hayth1Ross Brumett2Emil F. Khisamutdinov3Department of Chemistry, Ball State University, Muncie, IN 47306, USADepartment of Chemistry, Ball State University, Muncie, IN 47306, USADepartment of Chemistry, Ball State University, Muncie, IN 47306, USADepartment of Chemistry, Ball State University, Muncie, IN 47306, USANucleic acid-based therapeutics involves the conjugation of small molecule drugs to nucleic acid oligomers to surmount the challenge of solubility, and the inefficient delivery of these drug molecules into cells. “Click” chemistry has become popular conjugation approach due to its simplicity and high conjugation efficiency. However, the major drawback of the conjugation of oligonucleotides is the purification of the products, as traditionally used chromatography techniques are usually time-consuming and laborious, requiring copious quantities of materials. Herein, we introduce a simple and rapid purification methodology to separate the excess of unconjugated small molecules and toxic catalysts using a molecular weight cut-off (MWCO) centrifugation approach. As proof of concept, we deployed “click” chemistry to conjugate a Cy3-alkyne moiety to an azide-functionalized oligodeo-xynucleotide (ODN), as well as a coumarin azide to an alkyne-functionalized ODN. The calculated yields of the conjugated products were found to be 90.3 ± 0.4% and 86.0 ± 1.3% for the ODN-Cy3 and ODN-coumarin, respectively. Analysis of purified products by fluorescence spectroscopy and gel shift assays demonstrated a drastic amplitude of fluorescent intensity by multiple folds of the reporter molecules within DNA nanoparticles. This work is intended to demonstrate a small-scale, cost-effective, and robust approach to purifying ODN conjugates for nucleic acid nanotechnology applications.https://www.mdpi.com/1422-0067/24/5/4797bioconjugationclick chemistryODN conjugate purificationfluorescence reportersnucleic acid nanoparticles |
| spellingShingle | Erwin Doe Hannah L. Hayth Ross Brumett Emil F. Khisamutdinov Effective, Rapid, and Small-Scale Bioconjugation and Purification of “Clicked” Small-Molecule DNA Oligonucleotide for Nucleic Acid Nanoparticle Functionalization International Journal of Molecular Sciences bioconjugation click chemistry ODN conjugate purification fluorescence reporters nucleic acid nanoparticles |
| title | Effective, Rapid, and Small-Scale Bioconjugation and Purification of “Clicked” Small-Molecule DNA Oligonucleotide for Nucleic Acid Nanoparticle Functionalization |
| title_full | Effective, Rapid, and Small-Scale Bioconjugation and Purification of “Clicked” Small-Molecule DNA Oligonucleotide for Nucleic Acid Nanoparticle Functionalization |
| title_fullStr | Effective, Rapid, and Small-Scale Bioconjugation and Purification of “Clicked” Small-Molecule DNA Oligonucleotide for Nucleic Acid Nanoparticle Functionalization |
| title_full_unstemmed | Effective, Rapid, and Small-Scale Bioconjugation and Purification of “Clicked” Small-Molecule DNA Oligonucleotide for Nucleic Acid Nanoparticle Functionalization |
| title_short | Effective, Rapid, and Small-Scale Bioconjugation and Purification of “Clicked” Small-Molecule DNA Oligonucleotide for Nucleic Acid Nanoparticle Functionalization |
| title_sort | effective rapid and small scale bioconjugation and purification of clicked small molecule dna oligonucleotide for nucleic acid nanoparticle functionalization |
| topic | bioconjugation click chemistry ODN conjugate purification fluorescence reporters nucleic acid nanoparticles |
| url | https://www.mdpi.com/1422-0067/24/5/4797 |
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